The field of this invention is the diagnosis of galactosemia using nucleic acid-based techniques.
Galactosemia, an inherited metabolic disorder of milk sugar metabolism, is caused by certain mutations in the gene coding for the enzyme, galatose-1-phosphate uridyl transferase (GALT), which result in defective enzyme activity. This disorder is potentially fatal, so newborns throughout the U.S. and developed countries are screened for galactosemia, which when detected and treated early, saves lives and money. Evaluation of outcome in galactosemic patients began in the 1980s, and by 1990, an enigma was revealed (for example see Kornrower, GM, J. Inher. Metab. Dis., 1982, 5:96-104; Waggoner and Buist, 1993, Internat. Pediatr. 8:97-100). Although neonatal infants were screened and treated within days of life with a galactosexe2x80x94restricted diet, older children had unexpectedly poor outcomes. Dysfunctions included ovarian failure, verbal dyspraxia, growth and developmental delays, and neurological signs of cortical and extrapyramidal tract impairment. Since the GALT enzyme structure is highly conserved throughout evolution, the human GALT cDNA was cloned using probes homologous to known sequences in the bacterial enzyme. When the human cDNA and gene were sequenced (see Flach, Reichardt, and Elsas, 1990, Mol. Biol. Med. 7:365-369; and Leslie et al. 1992, Genomics 14:474-480), the first mutations in the GALT gene were associated with human galactosemia (Reichardt et al. 1991, Am. J. Hum. Genet. 49:860-867; Reichardt et al, 1992, Genomics 12:596-600) and relationships between the mutations and the resulting phenotype were being reported (Elsas et al. 1993, Internat Pediatr. 8:101). The Q188R mutation, first reported in 1991, is the most common mutation among galactosemic patients of Caucasian ethnicity (Reichert et al. 1991, Ibid.) and causes a complete loss in GALT activity. The S135L mutation is common among African-Americans (Lai et al., J. Pediatr 1996, 128:89-95), and results in severe reduction in GALT activity. The N314D mutation, also known as the Duarte allele (Elsas et al., 1994, Am J. Hum Genet 54:1030-1036), causes an instability in the GALT protein that leads to a 50% reduction in activity in homozygous patients. The K285N mutation is the second most common mutation among white galactosemic patients, and is the most prevalent among patients in southern Germany and Austria (Leslie et al., 1992, Ibid). Other common mutations identified in the white population are R148W and L195P (Reichart et al. 1992, Ibid).
These reports led to the realizations that different GALT mutations can result in different phenotypes and that therapy recommendations may vary depending on the specific mutation.
Methods currently used in newborn screening determine only the level of enzymatic activity or the accumulation of precursors. Results from current techniques are altered by the sampling parameters, such as ambient temperature and time of feeding relative to collection of sample. Also, they do not reveal the specific mutations that cause the change in enzyme activity. Certain mutations cause a mild disease, while others cause severe effects. Identifying the mutation is necessary to make a diagnosis, initiate appropriate therapy, estimate prognosis, and provide appropriate genetic counseling for the family.
There remains a need for a test with increased sensitivity and speificity for screening and diagnosis of galactosemia and asymptomatic carriers of galactosemia. There is a need for a test that can be run simultaneously on a large number of samples, such as in a micro titer plate format now commonly in use in clinical diagnostic laboratories. There is a need for a testing protocol that can quickly and specifically identify the majority of the alleles that can contribute to galactosemia in the human populations.
The claimed invention describes a nucleic acid-based method that can detect and specify the mutation involved in over 85% of individuals with some form of galactosemia rapidly and economically. Such a screen would be applicable to universal newborn screening and should supplement or completely replace current screening strategies. The screen can also be used to detect heterozygous mutations and the presence of multiple mutations in the GALT gene, which are necessary for understanding and counseling heritability and recurrence risks to family members.
The invention as claimed is a nucleic acid-based method that can detect and specify the mutation involved in the great majority of individuals with some form of galactosemia rapidly and economically. The method involves the detection of the mutation in amplified DNA from a patient utilizing probes that span the mutation. Six mutations are identified as being responsible for over 85% of the mutations in the United States population, and a detection kit is described for detecting these six mutations. The methods of this invention can also be used to determine the zygosity of the mutations and to detect the presence of multiple mutations in the GALT gene, both of which are necessary for understanding and counseling heritability and recurrence risks to family members.